The present invention relates to a mutein of a fibroblast growth factor (hereinafter also referred to as FGF) and a technique for producing the same.
Fibloblast growth factors (FGFs) are peptide growth factors, which include FGFs basic in isoelectric point (bFGFs) and FGFs acidic in isoelectric point (aFGFs). The whole amino acid sequences of these factors have both been revealed [F. Esch et al., Proc. Natl. Acad. Sci. U.S.A. 85, 6507 (1985) and K. A. Thomas et al., Proc. Natl. Acad. Sci. U.S.A. 82, 6409 (1985)].
It has been known that the FGF exhibits growth promoting action on mesoderm-derived cells such as 3T3 cells and vascular endothelial cells in vitro, and angiogenic action thereon in vivo [D. Gospodarowicz et al., Endocrine Reviews, 95 (1987)]. In particular, the angiogenic action of the FGF, together with its cell growth promoting action, is suggestive of a potential therapeutic application for traumas and burns, and as a preventive therapeutic medicine for thrombosis, arteriosclerosis, etc.
The FGFs naturally occur in extremely small quantities, and therefore, it was difficult to collect these. However, producing methods of the FGFs using genetic engineering techniques have recently been developed. The production methods using the genetic engineering techniques are reported in Biochem. Biophys. Res. Commun. 146, 470 (1987); Biotechnology, 5,960 (1987); The Journal of Biological Chemistry 263, 16471 (1988); The Journal of Biological Chemistry 263, 18452 (1988); The Journal of Biological Chemistry 263, 16297 (1988), and the like.
As to FGF muteins, a mutein in which Cys residue existing in the FGF is replaced with Ser is described in Biochem. Biophys. Res. Commun. 151, 701 (1988) and The Journal of Biological Chemistry 263, 18452 (1988).
The present inventors presumed that, by modifying the amino acid sequence to introduce a glycosylation site, the FGFs would be improved in stability, intercellular productivity and cell growth promoting activity per molecule, and provided with extracellular secretory activity, and, in addition, that its unidentified biological activities would be activated.
There are no data showing that a sugar chain is ligated to the naturally producing FGF. In general, the FGF is considered to be a peptide having no sugar chain. The FGF is a very unstable peptide and easily loses its activities, though it is not confirmed whether or not this is due to the above structure of the FGF. It is essential for the application of the FGFs to medicine and the like to improve stability of FGFs, particularly recombinant FGFs.
It has further been proved that a FGF gene has no clear leader sequence, and that most of the FGFs synthesized in cells remain in the cells and a very small amount of the FGFs or no FGFs are secreted outside the cells. No attempts have been successful till now to ligate a typical heterolognus leader sequence upstream from the FGF gene to produce and secrete the FGFs. In the production of the FGF by use of a genetic engineering, if the FGF can be secreted outside the cells, it is expected that subsequent purifying processes will be very simplified.